Primer and probe for detecting vibrio cholerae or vibrio mimicus and detection method using the same

a technology of vibrio cholerae and probes, which is applied in the field of primers and probes for detecting vibrio cholerae or vibrio mimicus, can solve the problems of inability to detect the strain to be detected with high accuracy, existing genetic detection methods, and inability to detect high-precision strains, etc., and achieve excellent amplification efficiency and high specificity. , the effect of high specificity

Inactive Publication Date: 2006-06-01
NICHIREI FOODSKK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0027] The use of information on specificity between phyletic group (FIGS. 2, 3, and 4) of the nucleotide sequences obtained in the present invention enables the design of probes having high specificity and gene amplification primers having high specificity and excellent amplification efficiency. Table 2 shows the sequences of PCR primers produced based on specific nucleotide information regarding Vibrio cholerae or Vibrio mimicus (FIGS. 2, 3, and 4) obtained in the present invention. TABLE 2Primers for specifically detecting V. choleraeand V. mimicusTargetgenePrimerSequenceLengthPositionDirectiongyrBCMgFgaaytctggcgtgtcgatcaag22258-279SenseCMgRcatrtagttgttcaaagtacgg22564-543AntisenserpoDCMrFgaycctaacgacatggaaacc21166-186SenseCMrRgtcacgaccaaattcattaac21420-400Antisense

Problems solved by technology

As described above, it has been impossible to detect with high accuracy Vibrio cholerae and Vibrio mimicus by the existing genetic detection methods.
The use of a nucleotide sequence of a single bacterial strain that is inferred to be a member of a bacterial population as a common sequence for such population or a sequence representing such population is very dangerous in terms of molecular evolutional characteristics of genes, which rapidly accumulate neutral mutations.
Specifically, there is concern that such use could cause misidentification such that a bacterial strain to be detected could not be detected because of inhibited amplification due to a slight mutation in a primer region.
There is also concern that a closely related strain not to be detected would be detected because of insufficient primer specificity.
In addition, as in the case of a gene that is transmitted horizontally at a high frequency (e.g., a toxin gene of Vibrio parahaemolyticus), a gene that exists independently of the phylogenetic line cannot be used.

Method used

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  • Primer and probe for detecting vibrio cholerae or vibrio mimicus and detection method using the same
  • Primer and probe for detecting vibrio cholerae or vibrio mimicus and detection method using the same
  • Primer and probe for detecting vibrio cholerae or vibrio mimicus and detection method using the same

Examples

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example 1

[0038] An example of using gene amplification primers (shown in Table 2) designed and obtained according to the present invention using regions specific to Vibrio cholerae and mimicus is shown. In addition, primers described in claim 5 (2) and (3) and claim 11 (2) and (7) correspond to CMgF, CMgR, CMrF, and CMrR in Table 2, respectively. PCR was carried out using chromosomal DNAs as templates extracted from test strains, AmpliTaq Gold (PE Applied Biosystems), and a total of 20 μl of reaction solution. Regarding thermal cycler conditions, after 10 minutes of heating at 95° C., 35 cycles of 94° C. for 1 minute, 1 minute of annealing (see Table 5 for annealing temperatures), and 72° C. for 1 minute were conducted, finally followed by 10 minutes of elongation reaction at 72° C. Samples obtained after reaction were subjected to 1% agarose gel electrophoresis, and then stained with ethidium bromide. The presence or the absence of amplified genes was confirmed under UV irradiation. Amplifi...

example 2

[0039] An example of using gene amplification primers (shown in Table 3 and Table 4) designed and obtained according to the present invention using regions specific to Vibrio cholerae or mimicus is shown. In addition, primers described in claim 19 (1), (3), (4), and (5) and claim 25 (1) and (14) are specific to Vibrio cholera. These primers are described in CF1, CF2, CR2, CR1, CrF1, and CrR1 in Table 3. Primers described in claim 33 (1), (3), (4), and (5) and claim 39 (7) and (13) are specific to Vibrio mimicus. These primers correspond to MF1, MF2, MR2, MR1, MrF1, and MrR1 in Table 4, respectively.

[0040] In a manner similar to Example 1, PCR was carried out using as templates chromosomal DNAs extracted from test strains (see Table 5 for annealing temperatures). In all cases, the use of Vibrio cholera-specific primers resulted in detection of amplification products only from Vibrio cholera and the use of Vibrio mimicus-specific primers resulted in detection of amplification product...

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Abstract

To produce high-performance specific gene amplification primers for detecting, quantifying, or identifying Vibrio cholerae and Vibrio mimicus, having low risk of misidentification and practically sufficient amplification efficiency and amplification specificity. We have determined partial nucleotide sequences of rpoD and gyrB genes of Vibrio cholerae, Vibrio mimicus, and closely related species, revealed their phylogenetic relationship, and then identified nucleotides characteristic of Vibrio cholerae and Vibrio mimicus, respectively. Thus, we have made it possible to design probes having high specificity and gene amplification primers having high specificity and excellent amplification efficiency, both of which contain the characteristic nucleotides.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for detecting, identifying, and counting Vibrio cholerae and Vibrio mimicus in food inspection, epidemiological environmental inspection, and clinical examination. BACKGROUND ART [0002]Vibrio cholerae produces a cholera toxin after oral infection and induces violent diarrhea and vomiting. In some cases, the infectious disease bacterium may cause affected patients to die from extreme dehydration. It has been thought that Vibrio cholerae, being toxic to humans, is only 01 cholerae that agglutinates by the use of an anti-O1 antibody. It has also been thought that Vibrio other than the bacterium is classified as NAG (O1-non-agglutinable)-Vibrio and is not toxic to humans. However, in the Bengal district in India in 1995, a new type of Vibrio cholerae having no O1 antigen that causes symptoms similar to those caused by conventional cholerae was isolated, revealing that the new Vibrio cholerae strain has a new O-antigen refe...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04C07K14/28C12N15/31
CPCC07K14/28C12Q1/689C12Q2600/156Y02A50/30
Inventor KOIZUMI, TAKESHINISHIYAMA, YOKOYAMAMOTO, SATOSHIFUKUYAMA, MASAFUMIFURUHATA, KATSUNORIOONAKA, KENJI
Owner NICHIREI FOODSKK
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